Preparation of reusable enzyme strips using alkylamine and arylamine glass beads affixed on plastic strips for urea determination

Abstract

<smarttagtype namespaceuri="urn:schemas-microsoft-com:office:smarttags" name="metricconverter"> Reusable strips of urease were prepared by covalently immobilizing it onto alkyl and arylamine glass beads, affixed on a plastic strip with a non-reactive adhesive. The immobilized urease retained its initial activity, prior to immobilization about 56% on alkylamine and 33.3% on arylamine. Maximum activity of alkylamine conjugated urease was attained at <i>p</i>H 6.5, 25°C for 1 min, while arylamine conjugated urease showed maximum activity at <i>p</i>H 7.0, 40^oC, 15 min. The <i>K</i>_m for urea was 4.65 and 16.66 mM, for alkyl and arylamine conjugated urease respectively. A method for determination of serum urea developed using these strips is based on measurement of NH₃ generated with Nessler’s reagent. The minimum detection limit permitted by the method was 10 and 5 mg/dL using alkyl and arylamine conjugated urease, respectively. Urea value in serum of healthy persons determined by this method, was in the range of 5-55 mg/dL with a mean of 26 mg/dL. The recovery of added urea was 82% for alkylamine and 85% for arylamine glass beads. A fair correlation (r=0.821) was found between urea values obtained by the standard urease kit and the present method. The alkylamine and arylamine conjugated urease stored at 4°C in 0.02 M potassium phosphate buffer <i>p</i>H 7.0 were reused 100 and 90 times respectively, during the span of one month. The method offers advantage of enormous ease in handling of immobilized urease.<b style=""> </b> </smarttagtype>